3,6-disubstituted-1,8-naphthalimides and methods for their production and use

ABSTRACT

Chemical compounds are provided that are novel 3,6-disubstituted-1,8-naphthalimides (I), as well as methods for their production, pharmaceutical compositions comprising the compounds, and methods of treatment using the compounds in dosage form. Compounds of the invention have pharmacological properties and are useful antimicrobial agents. ##STR1##

CROSS REFERENCE TO RELATED APPLICATION

This application is a division, of application Ser. No. 581,594, filedFeb. 21, 1984, which is a continuation-in-part of application Ser. No.481,122 filed Apr. 1, 1983, issued as U.S. Pat. No. 4,499,266 on Feb.12, 1985.

TECHNICAL FIELD

The invention relates to novel 3,6-disubstituted-1,8-naphthalimides, tomethods for their production, to pharmaceutical compositions comprisingthe compounds, and to methods of treatment using the compounds in dosageform. The compounds of the invention have pharmocological properties andare useful antimicrobial agents.

BACKGROUND OF THE INVENTION

N-substituted 3-nitro-1,8-phthalimides are known as described in theliterature, for example, U.S. Pat. No. 4,146,720, inter alia.

SUMMARY OF THE INVENTION

The invention in one aspect relates to3,6-disubstituted-1,8-naphthalimide compounds having in free base formthe structural formula I: ##STR2## and the pharmaceutically acceptablesalts thereof, where n is 2 or 3, R₁ and R₂ are H, lower alkyl, lowerhydroxyalkyl, pyrrolidinyl, morpholino, or piperidinyl, and Y is NO₂ orNH₂. The terms lower alkyl or hydroxyalkyl as used herein refer to suchgroups containing 1 to 6 alkyl carbon atoms.

The compounds of the invention form pharmaceutically acceptable saltswith both organic and inorganic acids. Examples of suitable acids forsalt formation are hydrochloric, sulfuric, phosphoric, acetic, citric,oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic,methanesulfonic, isethionic, lactic, gluconic, glucuronic, sulfamic,benzoic, tartaric, pamoic, an the like. The salts are prepared bycontacting the free base form with an equivalent amount of the desiredacid in the conventional manner. The free base forms may be regeneratedby treating the salt form with a base. For example, dilute aqueous basesolutions may be utilized. Dilute aqueous sodium hydroxide, potassiumcarbonate, ammonia, and sodium bicarbonate solutions are suitable forthis purpose. The free base forms differ from their respective saltforms somewhat in certain physical properties such as solubility inpolar solvents, but the salts are otherwise equivalent to theirrespective free base forms for purposes of the invention.

The compounds of the invention can exist in unsolvated as well assolvated forms, including hydrated forms. In general, the solvated formswith pharmaceutically acceptable solvents such as water, ethanol and thelike are equivalent to the unsolvated forms for purposes of theinvention.

The invention in another aspect relates to compounds having thestructural formula I, and the pharmaceutically acceptable salts thereof,that are most preferred for their pharmocological properties. Thesecompounds in free base form, have the following names:

N-[2-dimethylamino)ethyl]-3,6-dinitro-1,8-naphthalimide,

3,6-dinitro-N-[2-(pyrrolidinyl)ethyl]-1,8-naphthalimide,

N-[2-(diethylamino)ethyl]-3,6-dinitro-1,8-naphthalimide,

3,6-dinitro-N-[2-(morpholino)ethyl]-1,8-naphthalimide,

3,6-dinitro-N-[2-(piperidinyl)ethyl]-1,8-naphthalimide,

N-[2-dimethylamino)ethyl]-3,6-diamino-1,8-naphthalimide, and

3,6-diamino-N-[1-(piperidinyl)ethyl]-1,8-naphthalimide.

PROCESS FOR PREPARING THE COMPOUNDS

The invention in one process aspect comprises a process for preparingcompounds having the structural formula I: ##STR3## where Y is NO₂ byreacting 3,6-dinitro-1,8-naphthalic anhydride and an alkylenediaminehaving the structural formula H₂ N-(CH₂)_(n) -NR₁ R₂, preferably inexcess, under dehydration conditions and isolating the product in freebase form or pharmaceutically acceptable salt form; where n, R₁, and R₂have the above meaning. The reaction conditions are subject toconsiderable variation. The reaction is conveniently carried out in aninert solvent such as toluene, at room temperature for a short periodfollowed by reacting at reflux temperature for removal of water. Theproduct is preferably isolated in acid addition salt form obtained bytreating the free base in solution with a selected acid in solution suchas methanolic HC1. The starting materials are known compounds.

Purification of compounds or products obtained by methods of theinvention is accomplished in any suitable way, preferably by columnchromatography or crystallization.

The invention in another aspect comprises a process for preparingcompounds having the structural formula I above where Y is NH₂ bysubjecting a compound having the structural formula I where Y is NO₂ toreduction and isolating the product in free base form orpharmaceutically acceptable salt form, where n, R₁ and R₂ have the abovemeaning. The reduction is carried out by suitable means, preferablycatalytically by hydrogenation at room temperature using apalladium/charcoal catalyst in water, or chemically by reaction with tinmetal in an aqueous acidic medium at elevated temperature, preferably attemperatures in the range from about 90° to about 100° C.

The invention in its composition aspect relates to a pharmaceuticalcomposition for treating microbial infection comprising a compoundhaving structural formula I and the pharmaceutically acceptable saltsthereof in combination with a pharmaceutically acceptable carrier.

The invention in another aspect relates to a pharmaceutical compositionfor treating leukemia implantable in a lower mammal, comprising acompound having structural formula I and the pharmaceutically acceptablesalts thereof in combination with a pharmaceutically acceptable carrier.

The invention in another aspect relates to a pharmaceutical compositionfor treating solid tumors implantable in a lower mammal, comprising acompound having structural formula I and the pharmaceutically acceptablesalts thereof in combination with a pharmaceutically acceptable carrier.

The invention in another method aspect relates to a method for treatingmicrobial infections in a mammal which comprises administering asufficient amount of a compound having structural formula I and thepharmaceutically acceptable salts thereof in combination with apharmaceutically acceptable carrier.

The invention in another method aspect relates to a method for treatingimplantable leukemia in a lower mammal which comprises administering asufficient amount of compound having structural formula I and thepharmaceutically acceptable salts thereof in combination with apharmaceutically acceptable carrier, to a mammal in need thereof.

The invention in another method aspect relates to a method for treatingsolid tumors implantable in a lower mammal which comprises administeringa sufficient formula I and the pharmaceutically acceptable salts thereofin combination with a pharmaceutically acceptable carrier, to a mammalin need thereof.

PHYSICAL AND PHARMACOLOGICAL PROPERTIES OF THE COMPOUNDS

The compounds of the invention are useful as pharmacological agents forthe treatment of microbial infections, and for the treatment ofimplantable leukemia and solid tumors in lower warm-blooded animals. Theactivity of representative compounds of the invention was established bytest protocols described below.

TEST PROTOCOLS 1. In Vitro

One test protocol is the in vitro proliferating human colonadenocarcinoma (HCA) cell screen. In this test, HCT-8 cells (HCA cellline received from Yale University) are trypsinized using trypsin-EDTA.A single cell suspension is achieved by passing the cells through a 26gauge needle with a 20 cc syringe. A cell suspension is prepared usingRPMI 1640+10% FCS+50 ug/ml gentamicin sulfate with a cell concentrationof approximately 30,000 cells/ml. The cell suspension is dispensed inLinbro 24-well plates; 1 ml/well. The plates are incubated forapproximately 48 hours at 37 degrees C. in a 5% CO₂ atmosphere. At thistime test compounds are added in the appropriate concentration. Five ulof the 200 ug/ml stock solution is added to each well in a primary test.Ten ul of the appropriate dilution is added to each well for a titrationtest. The plates are reincubated an additional 60-65 hours at 37 degreesC. in a 5% CO₂ atmosphere. The cells are lysed using a mix of cationicsurfactant, glacial acetic acid and sodium chloride. Two ml of the lysedcell suspension from each well is added to 8 ml of diluent. The numberof nuclei is determined using a Coulter counter (ZBI model), and apercent growth for each drug concentration is calculated. From this, anID₅₀ (molar concentration of compound that results in 50% inhibition ofgrowth) is determined.

Another test protocol utilizes the solid tumor B16 melanoma in mice. Thetumor is inoculated by intraperitoneal injection. The test compounds areadministered intraperitoneally once daily for nine consecutive days atvarious doses following tumor inoculation. A ratio of survival time fortreated (T)/control (C) animals is calculated. A criterion for efficacyis a ratio T/C times 100 greater than or equal to 15. See Cancer TherapyReviews, 7, 167 (1980) and references cited therein for further detailsand interpretation of the test.

Another test protocol is the in vitro antibacterial/antifungal (ABF)test. Compounds are tested for antimicrobial activity in an agar-diskdiffusion assay, a standard microbiological technique for testingantibiotics. After incubation of each culture with a test compound, azone of inhibition is determined. The zone diameter (mm) of activecompounds ranges from a minimum of 13.5 mm to as high as 60 mm, with agreater diameter reflecting higher activity. For convenience, values arereported for the gram-negative bacterial species (Escherichia coli04863), two gram positive bacteria (Bacillus subtilis 04555 andStreptococcus faecalis 05045 utilizing AM-09 medium), and one mycelialfungus (Penicillium avellaneum M2988).

In another test protocol, growth-inhibitory effects are measured againstthe mouse tumor line L1210 in suspension cell culture. Cell cultureswere initiated at a density of 50,000 cells/ml in medium RPMI 1630supplemented with 10 percent fetal calf serum. Composition of thisculture medium and details of the culture procedure follow the publishedmethod (Journal of the National Cancer Institute 36, 405-415, 1966).Cultures are maintained at 37 degrees in stationary suspension cultureunder a 95 percent air +5 percent CO₂ atmosphere. Test compounds areadded to treated cultures at the time of initiation, and are presentcontinually. After 72 hours, 40-fold dilutions of drugtreated anduntreated control cultures are prepared in 0.9 percent NaCl solution,and cells are counted on an electronic particle counter. Thegrowth-inhibitory effects are expressed as ID₅₀ values, namely, testcompound inibitory dosages or concentrations required to decrease cellcount in treated cultures to 50 percent of the cell count of untreatedcontrol cultures.

2. In Vivo

Another test protocol is the in vivo lymphocytic leukemia P388 test. Theanimals used are either male or female CD₂ F₁ mice, six or seven animalsper test group. The tumor transplant is by intraperitoneal injection ofdilute ascitic fluid containing cells of lymphocytic leukemia P388. Thetest compounds are administered intraperitoneally once daily for fiveconsecutive days at various doses following tumor inoculation. Theanimals are weighed and survivors are recorded on a regular basis for 30days. A compound is designated "toxic" if, at a given dose, all animalsdied prior to four days after the first injection of drug. A ratio ofsurvival time for treated (T)/control (C) animals is calculated. Acriterion for efficacy is a ratio T/C times 100 greater than or equal to125. See Cancer Chemotherapy Reports, Part 3, 3, 1 (1972) for acomprehensive discussion of the protocol.

These test protocol procedures gave results listed in Tables 1, 2 and 3for representative compounds of the invention having the followingformula Ia ##STR4## where R has the meaning shown in the tables.

                  TABLE 1                                                         ______________________________________                                        Naphthylimide in vitro Activity                                               vs Human Colon Adenocarcinoma                                                 R                 ID.sub.50 Molar                                             ______________________________________                                        CH.sub.2 CH.sub.2 NEt.sub.2                                                                     9.6 × 10.sup.-7                                       CH.sub.2 CH.sub.2 N(CH.sub.2).sub.4                                                             1.8 × 10.sup.-7                                                         8.6 × 10.sup.-7                                       CH.sub.2 CH.sub.2 N(CH.sub.2).sub.5                                                             4.8 × 10.sup.-7                                       ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Naphthylimide Antimicrobial Activity                                                      Inhibition Zone Diameter mm                                                   (Dose mg/ml)                                                                            B. Sub- P. avel-                                        R             E. Coli tilis   laneum S. Faecalis                              ______________________________________                                        CH.sub.2 CH.sub.2 N(CH.sub.2).sub.4                                                         17(500) 16(100) 22(500)                                                                              18(500)                                   ##STR5##             22(100)  15(3000)                                                                            14(500)                                  CH.sub.2 CH.sub.2 N(CH.sub.2).sub.5                                                         16(500) 24(100) 26(500)                                                                              19(500)                                  ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Naphthylimide Antitumor Activity                                                           L1210 Leukemia                                                                In Vitro     Dose     T/C × 100                            R            ID.sub.50 Molar                                                                            mg/kg    (Percent)                                  ______________________________________                                                            P388 Leukemia                                                                 In Mice                                                   CH.sub.2 CH.sub.2 NMe.sub.2                                                                3.6 × 10.sup.-8                                                                      3.12     167                                        CH.sub.2 CH.sub.2 NEt                                                                      3.5 × 10.sup.-7                                                                      1.56     155                                        CH.sub.2 CH.sub.2 N(CH.sub.2).sub.4                                                        8.3 × 10.sup.-8                                             ##STR6##    1.1 × 10.sup.-6                                            CH.sub.2 CH.sub.2 N(CH.sub.2).sub.5                                                        2.9 × 10.sup.-7                                                                B16 Melanoma                                              CH.sub.2 CH.sub.2 NMe.sub.2                                                                         1.00     125                                            CH.sub.2 CH.sub.2 N(CH.sub.2).sub.5                                                                 12.5     135                                            Compounds of the invention having formula I where Y is                        NH.sub.2 typically have comparable activity.                                  ______________________________________                                    

PREPARATION OF PHARMACEUTICAL COMPOSITIONS

When being utilized as pharmacological agents, the compounds of theinvention can be prepared and administered in a wide variety of topical,oral, and parenteral dosage forms. It will be clear to those skilled inthe art that the following dosage forms may comprise as the activecomponent, one or more compounds of formula I, a correspondingpharmaceutically acceptable salt of any of said compounds, or a mixtureof such compounds and/or salts.

For preparing pharmaceutical compositions from the compounds describedby this invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets, and suppositories. Asolid carrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, or tablet disintegrating agents; it can also be anencapsulating material. In powders, the carrier is a finely dividedsolid which is in admixture with the finely divided active compound. Inthe tablet the active compound is mixed with carrier having thenecessary binding properties in suitable proportions and compacted inthe shape and size desired. The powders and tablets preferably containfrom 5 or 10 to about 70 percent of the active ingredient. Suitablesolid carriers are magnesium carbonate, magnesium stearate, talc, sugar,lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose,sodium carboxymethyl cellulose, a low melting wax, cocoa butter, and thelike. The term "preparation" is intended to include the formulation ofthe active compound with encapsulating material as carrier providing acapsule in which the active component (with or without other carriers)is surrounded by carrier, which is thus in association with it.Similarly, cachets are included. Tablets, powders, cachets, and capsulescan be used as solid dosage forms suitable for oral administration.

Liquid form preparations inlude solutions, suspensions, and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection. Liquid preparations can also be formulated insolution in aqueous polyethylene glycol solution. Aqueous solutionssuitable for oral use can be prepared by dissolving the active componentin water and adding suitable colorants, flavors, stabilizing, andthickening agents as desired. Aqueous suspensions suitable for oral usecan be made by dispersing the finely divided active component in waterwith viscous material, i.e., natural or synthetic gums, resins, methylcellulose, sodium carboxymethyl cellulose, and other well-knownsuspending agents.

Topical preparations include dusting powders, creams, lotions, gels, andsprays. These various topical preparations may be formulated bywell-known procedures. See for example Remington's PharmaceuticalSciences, Chapter 43, 14th Ed., Mack Publishing Co., Easton, Pa. 18042,USA.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself or it can be the appropriate number of any of thesepackaged forms.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from 50 mg to 500 mg according to the particularapplication and the potency of the active ingredient.

In therapeutic use as pharmacological agents the compounds utilized inthe pharmaceutical method of this invention are administered at theinitial dosage of about 0.1 mg to about 50 mg per kilogram. A dose rangeof about 0.5 mg to about 10 mg per kilogram is preferred. The dosages,however, may be varied depending upon the severity of the conditionbeing treated, and the compound being employed. Determination of theproper dosage for a particular situation is within the skill of the art.Generally, treatment is initiated with smaller dosages which are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day if desired.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compound as a free base orpharmaceutically acceptable salt can be prepared in water suitably mixedwith a surfactant such as hydroxypropylcellulose. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), N,N-dimethylacetamide,suitable mixtures thereof and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed bysterilization accomplished by filtering. Generally, dispersions areprepared by incorporating the various sterilized active ingredient intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofthe sterile powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and thefreeze-drying technique which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances iswell-known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions inunit dosage form for ease of administration and uniformity of dosage.Unit dosage forms used herein refers to physically discrete unitssuitable as unitary dosages for the mammalian subjects to be treated;each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the novelunit dosage forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active material andthe particular therapeutic effect to be achieved, and (b) the limitationinherent in the art of compounding such an active material for thetreatment of disease in living subjects having a diseased condition inwhich bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically-acceptable carrier in unit dosage form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from about 0.1 to about 500 mg, withfrom about 0.5 to about 250 mg being preferred. Expressed inproportions, the active compound is generally present in from about 0.1to about 500 mg/ml of carrier. In the case of compositions containingsupplementary active ingredients, the dosages are determined byreference to the usual dose and the manner of administration of the saidingredients. The daily parenteral doses for lower mammalian subjects tobe treated ranges from 0.1 mg/kg to 100 mg/kg. The preferred dailydosage range is 0.3 mg/kg to 10 mg/kg.

The invention and the best mode of practicing the same are illustratedby the following examples of preferred embodiments of selected compoundsand their preparation.

EXAMPLE 1 N-[2-(Dimethylamino)ethyl]-3,6,-dinitro-1,8-naphthalimide,Hydrochloride

A mixture of 14 g (0.048 mole) of 3,6-dinitro-1,8-naphthalic anhydrideand 450 ml of toluene was azeotropically refluxed for 90 min. Thesolution was cooled to room temperature. To this solution was addeddropwise 5.6 g (0.065 mole) of unsym-N,N-dimethylethylenediamine in 7 mlof toluene. The addition took 5 min. The mixture was stirred at roomtemperature for 20 min., then refluxed in an oil bath at 140 degrees C.for 2 hours azeotropically. Water (1.2 ml) was collected. The reactionmixture was filtered while hot. The filtrate was washed successivelywith water 2×100 ml), 5% NaHCO₃ (2×100 ml) and water again (2×200 ml),then dried over anhydrous Na₂ SO₄. To the dried filtrate was added amixture of 40 ml of methanolic HCl (2.33 m mole HCl per 1 ml of MeOH)and 200 ml of ether with stirring. The resulting precipitate wascollected by filtration, washed with ether (2×40 ml), and dried to give12.3 g (62% yield) of the product, m.p. 296-298 degrees. An analyticalsample was prepared by recrystallation from methanol, m.p. 298-300degrees. λ(max MeOH) 268 nm (log ε4.64), 320 (3.93), and 332 (3.95).

Anal. Calcd for C₁₆ H₁₄ N₄ O₆.HCl0.11/2H₂ O: C, 45.56; H, 4.30; N,13.20. Found: C, 45.60; H, 4.25; N, 13.20.

EXAMPLE 2 3,6-Dinitro-N-[2-(pyrrolidinyl)ethyl]-1,8-naphthalimideHydrochloride

A mixture of 5.8 g (0.02 mole) of 3,6-dinitro-1,8-naphthalic anhydridein 300 ml of toluene was azeotropically refluxed for 1 hour, then cooledto room temperature. To this solution was added dropwise 2.7 g (0.024mole) of N-(2-aminoethyl)pyrrolidine in 30 ml of toluene in 10 min. Thereaction mixture was stirred at room temperature for 1 hour and refluxedazeotropically for 1 hour to remove 0.4 ml of water. The resulting brownsolution was washed successively with water 3×100 ml), 5% Na₂ CO₃ (2×100ml), and again water (4×100 ml), then dried (Na₂ SO₄). The aqueouswashings were extracted with toluene (4×100 ml.) The latter washed withwater (3×80 ml), dried and combined with the organic solution. To thetoluene solution was added a mixture of 100 ml of ether and 30 ml ofmethanolic HCl (containing 2.33 mmole of HCl per 1 ml of MeOH). Themixture was stirred for 30 min. The resulting precipitate was collectedby filtration, washed with ether (3×50 ml), and dried to give 7.3 g (87%yield) of the product, m.p. 262-264 degrees. An analytical sample wasobtained by recrystallization from methanol as long needles, m.p.294-295 degrees. λ max (MeOH) 200 nm (log ε 4.32), 264 (4.53), 320(3.80), and 330 (3.83).

Anal. Calcd for C₁₈ H₁₆ N₄ O₆.HCl: C, 51.37. H, 4.07.N, 13.31. Found: C,51.30; H, 4.12; N, 13.30.

EXAMPLE 3 N-[2-(Diethylamino)ethyl]-3,6-dinitro-1.8-naphthalimideHydrochloride

This compound was prepared using the procedure of Example 1 from 5.8 g(0.02 mole) of 3,6-dinitro-1,8-naphthalic anhydride in 3000 ml oftoluene and 2.8 g (0.024 mole) of unsym-N,N-diethylethylenediamine in 40ml of toluene to give 6.8 g (82% yield) of the product, m.p. 235-242degrees. An analytical sample was prepared by recrystallization frommethanol as brown plates, m.p. 252-254 degrees. λ max (MeOH) 206 nm (logε4.37), 266 (4.56), 320 (3.86), and 332 (3.88).

Anal. Calcd for C₁₈ H₁₈ N₄ O₆.HCl: C, 51.13; H, 4.53; N, 13.25. Found:C, 50.90; H, 4.60; N, 13.36.

EXAMPLE 4 3,6-Dinitro-N-[2-(morpholino)ethyl]-1,8-naphthalimide

This compound was prepared using the procedure for Example 1 from 5.8 g(0.02 mole) of 3,6-dinitro-1,8-naphthalic anhydride in 300 ml of tolueneand 3.2 g (0.024 mole) of N-(2-aminoethyl)morpholine in 40 ml of tolueneto give 4.1 g (46% yield) of the product, m.p. 250-254 degrees. Ananalytical sample was prepared by recrystallization from methanol asbrown crystals, m.p. 275-276 degrees. λ max (MeOH) 202 nm (log ε4.38),265 (4.49), 318 (3.89), and 330 (3.89).

Anal. Calcd for C₁₈ H₁₆ N₄ O₇.HCl: C, 49.49; H, 3.92; N, 12.82 Found: C,49.60; H, 4.20; N, 12.68

EXAMPLE 5 3,6-Dinitro-N-[2-(piperidinyl)ethyl]-1,8-naphthalimide,Hydrochloride

This compound was prepared using the procedure for Example 1 from 4.3 g(0.015 mole) of 3,6-dinitronaphthalic anhydride in 250 ml of toluene and2.4 g (0.018 mole) of N-(2-aminoethyl)piperidine in 40 ml of toluene togive 4.8 g (73% yield) of the product, m.p. 265-268 degrees. Ananalytical sample was prepared by crystallization from a mixture ofmethanol and ether, m.p. 264-266 degrees. λ max (MeOH) 202 nm (log ε4.43), 266 (4.60), 320 (3.89) and 330 (3.92).

Anal. Calcd for C₁₉ H₁₈ N₄ O₆.HCl: C, 52.48; H, 4.40; N, 12.88. Found: C52.12; H, 4.46; N, 12.70.

Using the following unsym. alkylene diamine starting materials, thefollowing compounds are prepared by the above exemplified procedures:

    ______________________________________                                                          3,6-Dinitro-1,8-Naphthalimide                               Starting Material Product, as Free Base                                       ______________________________________                                        H.sub.2 NCH.sub.2 CH.sub.2 CH.sub.2 NMe.sub.2                                                   N[3-(dimethylamino)propyl]-                                 H.sub.2 NCH.sub.2 CH.sub.2 CH.sub.2 NEt.sub.2                                                   N[3-(Diethylamino)propyl]-                                  H.sub.2 NCH.sub.2 CH.sub.2 CH.sub.2 N(CH.sub.2).sub.4                                           N[3-(Pyrrolidinyl)propyl]-                                   ##STR7##         N[3-(Morpholino)propyl]-                                    H.sub.2 NCH.sub.2 CH.sub.2 CH.sub.2 N(CH.sub.2).sub.5                                           N[3-(Piperidinyl)propyl]-                                   ______________________________________                                    

EXAMPLE 6 N-(2-Dimethylaminoethyl)-3,6-diamino-1,8-naphthalimideHydrochloride

Method 1 (Chemical Reduction)

A mixture of 2.0 g (5 mmoles) of the hydrochloride salt ofN-(dimethylaminoethyl)-3,6-dinitro-1,8-naphthalimide, 5.9 g (5.0 mmoles)of 20 mesh tin metal in 60 ml of water and 20 ml of conc. HCl was heatedat 90°-100° C. for 2 hours with stirring. After cooling, the insolublesolid was removed by filtration and washed with 2×30 ml of hot water.The combined filtrate and washings were evaporated to dryness. Theresulting residue was extracted with hot methanol (8×50 ml). Themethanolic extracts were evaporated to dryness and the residue wastriturated with 50 ml of ether, filtered and the solid washed withether. The resulting hygroscopic solid was recrystallized from 150 ml ofethanol to give 0.4 g of the tin complex of the hydrochloride salt ofthe diamino compound, m.p. 190°-193°. Additional 0.5 g of the productwas obtained by concentration of the mother liquor. The total yield was30%.

λ max (MeOH): 250 nm (log ε4.66), 273 (4.31) and 435 (4.09).

Anal. Calcd for C₁₆ H₁₈ N₄ O₂.3HCl.SnCl2, C, 32.17; H 3.54; N. 9.38.Found: C, 32.28; H, 3.71; N. 8.90

Method 2 (Catalytic Reduction)

A mixture of 2.0 g (5 mmoles) of the hydrochloride salt ofN-(dimethylaminoethyl)-3,6-dinitro-1,8-naphthalimide and 350 mg of 10%palladium-on-charcoal in 120 ml of water was hydrogenated at roomtemperature under 40 lbs/in² of hydrogen for one hour. Theoreticalamount of hydrogen was absorbed. To the mixture was added 3 ml of conc.HCl. It was then filtered and the filtrate was evaporated to drynessunder reduced pressure. The residue was triturated with 30 ml ofabsolute ethanol and filtered. The resulting solid was washed with ether(5×10 ml) and dried to give 2.1 g (95% yield) of the diamino compound.Recrystallization from 150 ml of methanol gave 0.7 g (31.7% yield) ofthe purified product, mp. 295°-297°. λ max (MeOH): 250 nm (log ε 4.57),275 (4.25), 430 (3.96) and 445 (3.68). Anal. Calcd for C₁₆ H₁₈ N₄ O₂-2.7HCl: C, 48.43; H, 5.25; N, 14.12. Found C, 48.80; H, 5.21; N, 13.60.

EXAMPLE 7 N-(1-Piperidinylethyl)-3,6-diamino-1,8-naphthalimide,Hydrochloride

A mixture of 4.4 g (10 mmoles) ofN-(1-piperidinylethyl)-3,6-dinitro-1,8-naphthalimide, 0.7 g of 5%palladium-on-characoal in 180 ml of water was hydrogenated at roomtemperature under 40 lbs/cm² of hydrogen for 90 minutes. To the reducedmixture was added 6 ml of conc. HCl. The catalyst was removed byfiltration and washed with water (3×30 ml). The combined filtrate andwashings were evaporated to dryness under reduced pressure. The residuewas recrystallized from a mixture of ethanol and ether. The purifiedcrystals were collected by filtration, washed with ether, and dried togive 2.7 g mp 256°-258° (69% yield) of the hydrochloric salt of theproduct. λ max (MeOH): 250 nm (log ε 4.51); 275 (4.19); 430 (3.96); and445 (3.93). Anal. Calcd for C₁₉ H₂₂ N₄ O₂. 1.7HCl: C, 56.99; H, 5.96; N,14.00. Found: C, 57.10; H, 6.00; N, 14.30. Mass spec: M/e=338 (C₁₉ H₂₂N₄ O₂ =338).

EXAMPLE 8 Preparation of Intravenous Formulations

A solution of 12.5 g ofN-[2-(dimethylamino)ethyl]-3,6-dinitro-1,8-naphthalimide (fromExample 1) as the hydrochloride salt is prepared in 1 liter of water forinjection at room temperature with stirring. The solution is sterilefiltered into 500 5-ml vials, each of which contains 2 ml of solutioncontaining 25 mg of compound, and sealed under nitrogen.

Alternatively, after sterile filtration into vials, the water may beremoved by lyophilization, and the vials then sealed aseptically, toprovide a powder which is redissolved prior to injection.

Having thus described our invention, what we claim and desire by LettersPatent to secure are the following:

We claim:
 1. A method for treating transplantable leukemia in a lowermammal which comprises administering an antileukemically effectiveamount of a compound having in free base form the structural formula I##STR8## or a pharmaceutically acceptable salt thereof, where n is 2 or3, R₁ and R₂ are H, lower alkyl, lower hydroxyalkyl, pyrrolidinyl,morpholino, or piperidinyl, and Y is NO₂ or HN₂, in combination with apharmaceutically acceptable carrier, to a mammal in need thereof.